The importance of phosphatidylethanolamine (PE) in biology is multi-faceted. PE is typically the second most abundant phospholipid component in biological membranes and thus plays a fundamental role in cellular autonomy and subcellular compartmentalization. In addition, PE is a precursor for other major lipids and is critical for a diverse range of specific biological functions. In eukaryotes, PE synthesis can occur via four separate pathways one of which is performed by phosphatidylserine decarboxylase 1 which resides in the inner mitochondrial membrane. Intriguingly, even though there are four distinct pathways to make PE, deletion of phosphatidylserine decarboxylase 1 is embryonically lethal in mice. Not much is known about the phosphatidylserine decarboxylase 1 mechanism, most of the lipid trafficking steps required for this pathway remain obscure, and how PE produced in mitochondria supports mitochondrial function is incompletely resolved. The overarching goal of this application is to begin filling in the numerous gaps in our knowledge about this essential biosynthetic pathway. In the first specific aim, we will identify functionally important structural motifs in phosphatidylserine decarboxylase 1. The catalytically active form of phosphatidylserine decarboxylase 1 is generated by an autocatalytic event that generates a large membrane anchored ? subunit that non-covalently interacts with the enzymatically essential small ? subunit. How phosphatidylserine decarboxylase 1 achieves specificity is not known, but a potential substrate binding motif in the ? subunit has been identified by homology modeling. One goal of Aim 1 is to test the generated homology model and define the substrate binding motif. Another major effort in Aim 1 is to utilize chemical crosslinking to identify the interactio surface between the ? and ? subunits. The goal of this mapping exercise is to determine if the ?/? interaction is critical for phosphatidylserine decarboxylase 1 activity, and if so, how. Phosphatidylserine decarboxylase 1 is embedded in the mitochondrial inner membrane and it is known that PE produced outside of mitochondria is unable to compensate for the absence of PE made in mitochondria. In Aim 2, we will exploit a novel short-circuit strategy to obtain a more comprehensive understanding of mitochondrial PE metabolism. Specifically, we will determine the ability of PE produced in the outer membrane to access the inner membrane, molecularly characterize trafficking steps required for mitochondrial PE production, and pinpoint if the synthetic lethal interaction between the mitochondrial PE and cardiolipin biosynthetic pathways reflects a defect in an essential inner membrane or outer membrane function. By obtaining a more comprehensive understanding of mitochondrial PE metabolism, novel therapeutic targets may be identified for those diseases in which PE has been implicated, including Alzheimer's and prion disease, and for the numerous pathological states, including cancer, associated with derangements in cardiolipin metabolism.